DNA is an essential process in cell division. Elucidation of the factors that control DNA replication almost certainly will lead to an understanding of the factors that promote or limit normal and abnormal growth. Increased understanding of the factors that control abnormal cell growth such as that in cancer will have direct and immediate health-related impact, particularly on clinical strategies for the treatment of malignant growth. The purpose of this research plan is to determine the molecular mechanisms of DNA replication in the frog Xenopus laevis. In vitro DNA synthesizing systems will be reconstituted from highly purified Xenopus laevis DNA polymerases alpha, beta, gamma and delta, and precisely defined isolated DNA templates. Other potential DNA replication proteins will be used in an attempt to reconstitute an efficient chain elongation system. These proteins include helix destabilizing proteins, DNA topoisomerasses I and II, RNAse H, DNA ligase, DNA primase, nucleoplasmin, and (potentially) others such as DNA helicases. The newly discovered Xenopus laevis DNA primase and linear concatenation factor will be characterized. Initiation of Xenopus laevis DNA replication will also be investigated by microinjection into eggs of supercoiled plasmid DNA molecules containing defined fragments of Xenopus laevis DNA, by characterization of DNA synthesis on D-loop structures (possible initiation intermediates) and by characterization of initiation factors. Finally, electron microscopy will be used to delineate the molecular mechanisms of initiation and chain elongation. The predominant methodologies used are nucleic acid enzymology, electrophoresis, sedimentation, microinjecton, and electron microscopy.